IL-6-GP130 signaling protects human hepatocytes against lipid droplet accumulation in humanized liver models.

Liver steatosis is an increasing health issue with few therapeutic options, partly because of a paucity of experimental models. In humanized liver rodent models, abnormal lipid accumulation in transplanted human hepatocytes occurs spontaneously. Here, we demonstrate that this abnormality is associated with compromised interleukin-6 (IL-6)-glycoprotein 130 (GP130) signaling in human hepatocytes because of incompatibility between host rodent IL-6 and human IL-6 receptor (IL-6R) on donor hepatocytes. Restoration of hepatic IL-6-GP130 signaling, through ectopic expression of rodent IL-6R, constitutive activation of GP130 in human hepatocytes, or humanization of an Il6 allele in recipient mice, substantially reduced hepatosteatosis. Notably, providing human Kupffer cells via hematopoietic stem cell engraftment in humanized liver mice also corrected the abnormality. Our observations suggest an important role of IL-6-GP130 pathway in regulating lipid accumulation in hepatocytes and not only provide a method to improve humanized liver models but also suggest therapeutic potential for manipulating GP130 signaling in human liver steatosis.

Defining the Activated Fibroblast Population in Lung Fibrosis Using Single-Cell Sequencing.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disorder driven by unrelenting extracellular matrix deposition. Fibroblasts are recognized as the central mediators of extracellular matrix production in IPF; however, the characteristics of the underlying fibroblast cell populations in IPF remain poorly understood. Here, we use an unbiased single-cell RNA sequencing analysis of a bleomycin-induced pulmonary fibrosis model to characterize molecular responses to fibrotic injury. Lung cells were isolated on Day 11 to capture emerging fibrosis and gene expression was analyzed by three complementary techniques, which, together, generated a 49-gene signature that defined an activated subpopulation of fibroblasts. However, none of the identified genes were specific to the activated cells or to the disease setting, implying that the activated fibroblasts are not uniquely defined, but exhibit a similar, yet amplified, gene expression pattern to control cells. Our findings have important implications for fibrosis research, including: 1) defining myofibroblasts with any single marker will fail to capture much of the underlying biology; 2) fibroblast activation is poorly correlated with expression of transforming growth factor-ß pathway genes; 3) single-cell analysis provides insight into the mechanism of action of effective therapies (nintedanib); 4) early events in lung fibrosis need not involve significant changes in fibroblast number; populations that do increase in number, such as macrophages, dendritic cells, and proliferating myeloid cells, may merit closer examination for their role in pathogenesis.

Cooperation between both Wnt/{beta}-catenin and PTEN/PI3K/Akt signaling promotes primitive hematopoietic stem cell self-renewal and expansion.

Although self-renewal is the central property of stem cells, the underlying mechanism remains inadequately defined. Using a hematopoietic stem and progenitor cell (HSPC)-specific conditional induction line, we generated a compound genetic model bearing both Pten deletion and ß-catenin activation. These double mutant mice exhibit a novel phenotype, including expansion of phenotypic long-term hematopoietic stem cells (LT-HSCs) without extensive differentiation. Unexpectedly, constitutive activation of ß-catenin alone results in apoptosis of HSCs. However, together, the Wnt/ß-catenin and PTEN/PI3k/Akt pathways interact to drive phenotypic LT-HSC expansion by inducing proliferation while simultaneously inhibiting apoptosis and blocking differentiation, demonstrating the necessity of complementary cooperation between the two pathways in promoting self-renewal. Mechanistically, ß-catenin activation reduces multiple differentiation-inducing transcription factors, blocking differentiation partially through up-regulation of Inhibitor of differentiation 2 (Id2). In double mutants, loss of Pten enhances the HSC anti-apoptotic factor Mcl-1. All of these contribute in a complementary way to HSC self-renewal and expansion. While permanent, genetic alteration of both pathways in double mutant mice leads to expansion of phenotypic HSCs, these HSCs cannot function due to blocked differentiation. We developed a pharmacological approach to expand normal, functional HSCs in culture using factors that reversibly activate both Wnt/ß-catenin and PI3K/Akt signaling simultaneously. We show for the first time that activation of either single pathway is insufficient to expand primitive HSCs, but in combination, both pathways drive self-renewal and expansion of HSCs with long-term functional capacity.

Tumour-initiating cells: challenges and opportunities for anticancer drug discovery.

The hypothesis that cancer is driven by tumour-initiating cells (popularly known as cancer stem cells) has recently attracted a great deal of attention, owing to the promise of a novel cellular target for the treatment of haematopoietic and solid malignancies. Furthermore, it seems that tumour-initiating cells might be resistant to many conventional cancer therapies, which might explain the limitations of these agents in curing human malignancies. Although much work is still needed to identify and characterize tumour-initiating cells, efforts are now being directed towards identifying therapeutic strategies that could target these cells. This Review considers recent advances in the cancer stem cell field, focusing on the challenges and opportunities for anticancer drug discovery.

Detection of functional haematopoietic stem cell niche using real-time imaging.

Haematopoietic stem cell (HSC) niches, although proposed decades ago, have only recently been identified as separate osteoblastic and vascular microenvironments. Their interrelationships and interactions with HSCs in vivo remain largely unknown. Here we report the use of a newly developed ex vivo real-time imaging technology and immunoassaying to trace the homing of purified green-fluorescent-protein-expressing (GFP(+)) HSCs. We found that transplanted HSCs tended to home to the endosteum (an inner bone surface) in irradiated mice, but were randomly distributed and unstable in non-irradiated mice. Moreover, GFP(+) HSCs were more frequently detected in the trabecular bone area compared with compact bone area, and this was validated by live imaging bioluminescence driven by the stem-cell-leukaemia (Scl) promoter-enhancer. HSCs home to bone marrow through the vascular system. We found that the endosteum is well vascularized and that vasculature is frequently localized near N-cadherin(+) pre-osteoblastic cells, a known niche component. By monitoring individual HSC behaviour using real-time imaging, we found that a portion of the homed HSCs underwent active division in the irradiated mice, coinciding with their expansion as measured by flow assay. Thus, in contrast to central marrow, the endosteum formed a special zone, which normally maintains HSCs but promotes their expansion in response to bone marrow damage.

N-cadherin expression level distinguishes reserved versus primed states of hematopoietic stem cells.

Osteoblasts expressing the homophilic adhesion molecule N-cadherin form a hematopoietic stem cell (HSC) niche. Therefore, we examined how N-cadherin expression in HSCs relates to their function. We found that bone marrow (BM) cells highly expressing N-cadherin (N-cadherin(hi)) are not stem cells, being largely devoid of a Lineage(-)Sca1(+)cKit(+) population and unable to reconstitute hematopoietic lineages in irradiated recipient mice. Instead, long-term HSCs form distinct populations expressing N-cadherin at intermediate (N-cadherin(int)) or low (N-cadherin(lo)) levels. The minority N-cadherin(lo) population can robustly reconstitute the hematopoietic system, express genes that may prime them to mobilize, and predominate among HSCs mobilized from BM to spleen. The larger N-cadherin(int) population performs poorly in reconstitution assays when freshly isolated but improves in response to overnight in vitro culture. Their expression profile and lower cell-cycle entry rate suggest N-cadherin(int) cells are being held in reserve. Thus, differential N-cadherin expression reflects functional distinctions between two HSC subpopulations.

PTEN-deficient intestinal stem cells initiate intestinal polyposis.

Intestinal polyposis, a precancerous neoplasia, results primarily from an abnormal increase in the number of crypts, which contain intestinal stem cells (ISCs). In mice, widespread deletion of the tumor suppressor Phosphatase and tensin homolog (PTEN) generates hamartomatous intestinal polyps with epithelial and stromal involvement. Using this model, we have established the relationship between stem cells and polyp and tumor formation. PTEN helps govern the proliferation rate and number of ISCs and loss of PTEN results in an excess of ISCs. In PTEN-deficient mice, excess ISCs initiate de novo crypt formation and crypt fission, recapitulating crypt production in fetal and neonatal intestine. The PTEN-Akt pathway probably governs stem cell activation by helping control nuclear localization of the Wnt pathway effector beta-catenin. Akt phosphorylates beta-catenin at Ser552, resulting in a nuclear-localized form in ISCs. Our observations show that intestinal polyposis is initiated by PTEN-deficient ISCs that undergo excessive proliferation driven by Akt activation and nuclear localization of beta-catenin.

PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention.

Haematopoietic stem cells (HSCs) must achieve a balance between quiescence and activation that fulfils immediate demands for haematopoiesis without compromising long-term stem cell maintenance, yet little is known about the molecular events governing this balance. Phosphatase and tensin homologue (PTEN) functions as a negative regulator of the phosphatidylinositol-3-OH kinase (PI(3)K)-Akt pathway, which has crucial roles in cell proliferation, survival, differentiation and migration. Here we show that inactivation of PTEN in bone marrow HSCs causes their short-term expansion, but long-term decline, primarily owing to an enhanced level of HSC activation. PTEN-deficient HSCs engraft normally in recipient mice, but have an impaired ability to sustain haematopoietic reconstitution, reflecting the dysregulation of their cell cycle and decreased retention in the bone marrow niche. Mice with PTEN-mutant bone marrow also have an increased representation of myeloid and T-lymphoid lineages and develop myeloproliferative disorder (MPD). Notably, the cell populations that expand in PTEN mutants match those that become dominant in the acute myeloid/lymphoid leukaemia that develops in the later stages of MPD. Thus, PTEN has essential roles in restricting the activation of HSCs, in lineage fate determination, and in the prevention of leukaemogenesis.

Characterization of Nfatc1 regulation identifies an enhancer required for gene expression that is specific to pro-valve endocardial cells in the developing heart.

Nfatc1 is an endocardial transcription factor required for development of cardiac valves. Herein, we describe identification and characterization of a tissue-specific enhancer in the first intron of murine Nfatc1 that activates a heterogenic promoter and directs gene expression in a subpopulation of endocardial cells of the developing heart: the pro-valve endocardial cells. This enhancer activity begins on embryonic day (E) 8.5 in endocardial cells at the ventricular end of the atrioventricular canal, intensifies and extends from E9.5 to E11.5 in endocardium along the atrioventricular canal and outflow tract. By E12.5, the enhancer activity is accentuated in endocardial cells of forming valves. Sequential deletion analysis identified that a 250 bp DNA fragment at the 3' end of the intron 1 is required for endocardial-specific activity. This region contains two short conserved sequences hosting a cluster of binding sites for transcription factors, including Nfat and Hox proteins. Electrophoresis mobility shift and chromatin immunoprecipitation assays demonstrated binding of Nfatc1 to the Nfat sites, and inactivation of Nfatc1 downregulated the enhancer activity in pro-valve endocardial cells. By contrast, mutation of the Hox site abolished its specificity, allowing gene expression in non pro-valve endocardium and extracardiac vasculature. Thus, autoregulation of Nfatc1 is required for maintaining high Nfatc1 expression in pro-valve endocardial cells, while suppression through the Hox site prevents its expression outside pro-valve endocardial cells during valve development. Our data demonstrate the first autonomous cell-specific enhancer for pro-valve endocardial cells and delineate a unique transcriptional mechanism that regulates endocardial Nfatc1 expression within developing cardiac valves.

Regulation of Fgf10 gene expression in the prostate: identification of transforming growth factor-beta1 and promoter elements.

Fibroblast growth factor 10 (FGF10) is a mesenchymal paracrine-acting factor that plays a key role in the organogenesis of the prostate, and Fgf10 transcripts exhibit a highly restricted expression pattern within prostatic mesenchyme. To study the regulation of Fgf10 we have used organ rudiments grown in vitro as well as a primary stromal cell system derived from the ventral mesenchymal pad (VMP), a condensed area of mesenchyme known to induce prostatic organogenesis. Characterization of VMP cells (VMPCs) showed that they retained expression of AR as well as transcripts for FGF10 and TGFbeta1, -2, and -3. We propose that VMPCs are a good model of specialized mesenchyme involved in prostatic organogenesis and are distinct from general urogenital sinus mesenchyme/stroma. Treatment of VMPCs with TGFbeta1 resulted in a rapid and transient decrease in Fgf10 transcript levels, which were reduced 9-fold at 3 h. TGFbeta1 also inhibited Fgf10 expression in VMP organ rudiments grown in vitro. To further analyze Fgf10 regulation, 6 kb of mouse genomic sequence 5' to the translation start site was characterized by promoter analysis. Deletion analysis of the Fgf10 promoter in VMPCs identified a region of the promoter that mediated a significant proportion of promoter activity as well as mediating promoter down-regulation by TGFbeta1. This element was located between nucleotides -182 and -172 and contained a consensus Sp1 binding site. Taken together, our data suggest that TGFbeta1 is a regulator of Fgf10 expression in prostatic mesenchyme and that a proximal element within the Fgf10 promoter plays an important role in its regulation and expression.